DNA encoding for a disease resistance gene from common bean and methods of use

ABSTRACT

The present invention provides novel purified and isolated nucleic acid sequences associated with disease resistance and tolerance in plants. Methods of using the nucleic acid sequences to confer disease resistance and tolerance to plants are also provided.

[0001] Work on this invention was sponsored in part by United StatesAgency for International Development Grant DAN 1310-G-SS-6008-00. TheGovernment may have certain rights in the invention.

FIELD OF THE INVENTION

[0002] The invention relates generally to disease resistance genes fromplants and more particularly to anthracnose resistance genes fromlegumes.

BACKGROUND OF THE INVENTION

[0003] Plants can be damaged by a wide variety of pathogenic organismsincluding viruses, bacteria, fungi and nematodes. Annual crop losses dueto these pathogens is in the billions of dollars. Synthetic pesticidesare one form of defense against pathogens and each year thousands oftons of such chemicals are applied to crops. However, there are concernsregarding the short term and long term environmental damage due tochemical pesticides use, as well as inherent risks to human health.

[0004] Plants also contain their own innate mechanisms of defenseagainst pathogenic organisms. Natural variation for resistance to plantpathogens has been identified by plant breeders and pathologists andbred into many crop plants. These natural disease resistance genes oftenprovide high levels of resistance to pathogens and represent the mosteconomical and environmentally friendly form of crop protection.

[0005] Genetic resistance is the most efficient way to controlanthracnose, the disease caused by the fungus Colletotrichumlindemuthianum, in common bean (Phaseolus vulgaris L.). The high geneticvariability observed in the pathogen population (Balardin, R. S. et al.,Phytopathology 87:1184-1191 (1997)) is associated with differentresistance genes present in the host (Balardin, R. S. et al., J. Amer.Sco. Hort. Sci. 123(6):1038-1047 (1998)). Seven independent dominantdisease resistance genes (Co-1 to Co-7) controlling anthracnose in beanhave been described (Balardin, R. S. et al., Phytopathology 87:1184-1191(1997)). Each of these genes confers resistance to certain races of thepathogen strongly suggesting that resistance to anthracnose in commonbean follows the gene-for-gene theory (Flor, H. H., Phytopathology45:680-685 (1947)). Certain resistance genes are more effective thanothers in controlling multiple races of the pathogen (Balardin, R. S. etal., J. Amer. Sco. Hort. Sci. 123(6):1038-1047 (1998)).

[0006] The bean breeding line SEL 1308 derived from the highly resistantdifferential cultivar G2333, is known to possess the single dominantCo-42 gene for anthracnose resistance (Young, R. A. et al., Theor. Appl.Genet. 96:87-94 (1998)). When inoculated with 34 selected races of C.lindemuthianum chosen to represent a diverse sample of the pathogenpopulation, SEL 1308 demonstrated a resistance index (RI) of 97%(Balardin, R. S. et al., J. Amer. Sco. Hort. Sci. 123(6):1038-1047(1998)). The only cultivar with a higher RI (100%) was G2333 known topossess the combination of three independent resistance genes, Co-42,Co-5, and Co-7 (Young, R. A. et al., Theor. Appl. Genet. 96:87-94(1998)). This three-gene combination confers resistance to all describedraces of the pathogen (Pastor-Corrales, M. A. et al., Plant Dis.78:959-962 (1994)). Among the reported resistance genes, the Co-42 genein SEL 1308 exhibits the broadest-based resistance in common bean(Balardin, R. S. et al., J. Amer. Sco. Hort. Sci. 123(6):1038-1047(1998)).

[0007] Although plants can be bred for disease resistance traits, thiscan be a long and tedious process. A conventional plant breeding programrequires as much as ten years to develop a new variety. Furthermore,once a new variety is introduced, it may not prove resistant to newforms of the pathogen it was selected to be resistant to. For example,new races of C. lindemuthianum pathogenic to current commercialvarieties of dry bean have been identified (Balardin, R. S. et al.,Plant Dis. 80:712 (1996); Kelly, J. D. et al., Plant Disease 78:892-894(1994)). Sources of resistance to anthracnose in adapted commercial beanvarieties grown in the U.S. are ineffective against these races (Kelly,J. D. et al., Plant Disease 78:892-894 (1994)), whereas new resistancesources in Mexican germplasm offers an effective solution for thecontrol of anthracnose (Pastor-Corrales, M. A. et al., Plant Dis.78:959-962 (1994)). For instance, resistance in the Mexican landracevariety G 2333 to 450 races of anthracnose is conditioned by acombination of three independent resistance genes (Co42, Co-5, Co-7;Pastor-Corrales, M. A. et al., Plant Dis. 78:959-962 (1994); Young, R.A. et al., Crop Sci. 37:940-946 (1997)).

[0008] It would thus be desirable to provide disease resistant genesfrom legumes. No disease resistance gene has been cloned from the beanspecies P. vulgaris or any related legume species. It would further bedesirable to provide genes associated with resistance to anthracnose.The Co-42 gene is a valuable candidate gene for molecular cloning due toits broad resistance and availability of a tightly linked marker (Young,R. A. et al., Theor. Appl. Genet. 96:87-94 (1998)). In common bean andsoybean, resistance genes analogs (RGAs) have been identified usingprimers specific to conserved regions of known resistance genes andmapped close to known disease resistance locus (Kanazin, V. et al., PNAS(USA) 93:11746-11750 (1996); Yu, Y. G. et al., PNAS (USA) 93:11751-17756(1996); Geffroy, V. et al., Theor. Appl. Genet. 96:494-502 (1998);Rivkin, M. I. et al., Genome 42:41-47 (1999)). Mapping of RGAs has beenthe only approach used to isolate known resistance gene sequences fromcommon bean. RGAs, however, are not always closely associated with aresistance phenotype and may be loosely linked to known resistance locuslimiting their value in chromosome walking to the gene. Additionalapproaches, such as map-based cloning are still needed to isolateresistance gene candidates in crop species. It would also be desirableto provide legume plants with disease resistance, preferably toanthracnose. It would be further desirable to transform plants usinganthracnose resistant genes to produce disease resistant plants.

SUMMARY OF THE INVENTION

[0009] The present invention provides novel purified and isolatednucleic acid sequences associated with disease resistance in common bean(Phaseolus vulgaris L.). The DNA for COK-4 isolated from strain SEL 1308is set forth in SEQ ID NO: 1 while the DNA for COK-4 isolated from P.vulgaris Black Magic is set forth in SEQ ID NO: 2. The deduced aminoacid sequence of COK-4 is also provided and set forth in SEQ ID NO: 3.The predicted COK-4 protein contains a serine-threonine kinase domainwith a highly hydrophobic membrane-spanning region.

[0010] Methods for making and using the DNA's encoding for COK-4 arealso provided. For example, COK-4 may be used to provide plants withdisease resistance, preferably to anthracnose. Vectors containing theDNA, transgenic plants and other organisms, e.g., E. coli, transfectedwith said vectors, as well as seeds from said plants, are also providedby the present invention. Additional objects, advantages, and featuresof the present invention will become apparent from the followingdescription and appended claims, taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The various advantages of the present invention will becomeapparent to one skilled in the art by reading the followingspecification and subjoined claims and by referencing the followingdrawings in which:

[0012]FIG. 1A is a schematic of the linkage map of the Co-42 locusshowing the position of the molecular markers;

[0013]FIG. 1B is a schematic of the genomic region containing the Co-42locus showing the contig developed from overlapping genomic clones;

[0014]FIG. 1C is the amino acid sequence for COK4 (SEQ ID NO: 3);

[0015]FIG. 2 is a comparison of the COK-4 and other reported plantserine-threonine kinases amino acid sequences (SEQ ID NOS: 3-9);

[0016] FIGS. 3A-3D are photographs of agarose gels showing restrictionanalysis of COK-4 amplified in several genotypes;

[0017] FIGS. 4A-4B show a comparison of the DNAs of COK-4 homologues(SEQ ID NOS: 1, 2 and 4).

[0018]FIG. 5 is an autoradiograph of a Southern blot of Eco RI digestedgenomic DNA from bean cultivar and a BAC clone hybridized with the COK-4gene; and

[0019]FIG. 6 is a photograph of an agarose gel showing the DNAamplification of six bean cultivars with OBB14 and OH181150 RAPDmarkers;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention provides novel isolated nucleic acidsequences conferring disease resistance to plants. The DNA sequencesencoding for COK-4 and conferring disease resistance isolated fromPhaseolus vulgaris L. are set forth in SEQ ID NO: 1 (strain SEL 1308)and SEQ ID NO: 2 (strain TO). The deduced amino acid sequence isprovided in SEQ ID NO: 3. The COK-4 protein has a serine-threoninekinase domain, which includes a protein kinase ATP-binding regionsignature (amino acids 53 to 79 of SEQ ID NO: 3), a primarytransmembrane domain (amino acids 202 to 224 of SEQ ID NO: 3; FIG. 1C),putative sites for N-myristoylation and N-glycosylation, and a cAMP- andcGMP-dependent protein kinase site (amino acids 41 to 44 of SEQ ID NO:3; FIG. 1C).

[0021] In one embodiment, the COK-4 nucleic acid sequences capable ofconferring disease resistance or tolerance to plants can bedistinguished from non-resistance conferring sequences by restrictionmapping. For example, restriction patterns were polymorphic among threebean cultivars known to possess different alleles at the CO-4 locus.Perfect co-segregation between restriction patterns and diseaseresistance phenotype was observed in 1350 F3 individuals. Preferably, aCOK-4 nucleotide sequence capable of conferring disease resistance toplant will have no more than two restriction sites for restrictionenzymes Kpn I, Mse I or Msp I, whereas sequences associated withnon-resistance to disease will have at least two restriction sites. Forexample, the nucleotide sequence of SEQ ID NO: 1 has one restrictionsite for Kpn I, Mse I or Msp I (Lane 2, FIGS. 3B-3D). Likewise, thenucleotide sequence of SEQ ID NO: 2 has one restriction site for Kpn I,Mse I, and Msp I (Lane 6, FIGS. 3B-3D). In contrast, the COK-4nucleotide sequence associated with non-resistance to plant diseases(SEQ ID NO: 4) has at least two restriction sites for Kpn I and threesites for Mse I and Msp I (Lane 7, FIGS. 3B-3D).

[0022] In another embodiment the sequences of the present inventionconferring disease resistance or tolerance to a plant can beincorporated in plant or bacteria cells using conventional recombinantDNA technology. Generally this involves inserting the DNA molecule intoan expression system, wherein the DNA molecule is inserted into theexpression system or vector in proper orientation and correct readingframe. The vector contains the necessary elements for the transcriptionand translation of the inserted protein-coding sequences. A large numberof vector systems known in the art can be used, such as, but not limitedto, plasmids, bacteriophage virus or other modified viruses. The DNAsequences are clones into the vector using standard cloning proceduresin the art.

[0023] The method of conferring disease resistance or tolerance to aplant includes the steps of introducing an expression vector comprisingcDNA encoding a COK-4 protein associated with disease resistance or afunctional mutant thereof, operably linked to a promoter functional in aplant cell into the cells of plant tissue and expressing the encodingprotein in an amount effective to render the plant tissue resistant ortolerant to disease. The choice of promoter will depend on the host cellsystem into which the gene is to be incorporated and the amount ofprotein expression desired. The level of expression can be increased byeither combining the cDNA with a promoter that provides for a high levelof expression, or by introducing multiple copies into the cell so thatmultiple copies are integrated into the genome of transformed plantcells. Once transferred cells exhibiting disease resistance or toleranceare obtained, transgenic plants and seeds can then be regeneratedtherefrom, and evaluated for stability of the inheritance of the diseaseresistance or tolerance trait.

[0024] Alternatively, seeds, seedlings or plants can be transformeddirectly with the nucleic acid sequences and expression vectors of thepresent invention. The most common methods for transforming plants are,by way of non-limiting example, Agrobacterium-mediated gene delivery,microprojectile bombardment, free DNA delivery to protoplasts andelectrophoretic transformation. Preferably, the low-voltagetransformation method described in U.S. Ser. No. 09/206,852(incorporated by reference) is used to transform plants using thenucleic acid sequences of the present invention. This method utilizeslow amperage current to deliver DNA into the meristematic cells andtissue of plants and plant seedlings.

[0025] A COK-4 nucleotide sequence associated with disease resistancemay thus be fused to a gene or fragment thereof, which allows it to beexpressed in a plant cell. The COK-4 nucleotide sequence in combinationwith the gene or gene fragment, is referred to as an “expression vector”herein. It will be appreciated that the expression vectors of thepresent invention may contain any regulatory elements necessary andknown to those skilled in the art for expression of COK-4. For example,such vectors may contain, but are not limited to, sequences such aspromoters, operators and regulators, which are necessary for and/or mayenhance the expression of COK-4.

[0026] In another embodiment, the nucleic acid sequences of the presentinvention are used to genetically engineer plants to confer diseaseresistance or tolerance. Preferably, the plants genetically altered withthe nucleic acid sequences of the present invention are of theLeguminosae family. This family includes, but is not limited to, peas,beans, chickpeas and peanuts. More preferably, the plants are of thegenus Phaseolus and most preferably are from the common bean group,Phaseolus vulgaris. Non-limiting examples of common bean are green bean,kidney bean, snap bean, haricot, runner bean, wax bean, black bean andpinto bean.

[0027] In a further embodiment, the nucleic acid sequences of thepresent invention are used to genetically engineer plants which areresistant or tolerant to fungal diseases. Non-limiting examples of plantfungal diseases are rust and root rot. Preferably, the geneticallyengineered plants of the present invention will be resistant or tolerantto the fungal disease Collectotrichum lindemuthianum, commonly known asanthracnose.

[0028] As referred to herein, the term “cDNA” is meant a nucleic acid,either naturally occurring or synthetic, which encodes a proteinproduct. The term “nucleic acid” is intended to mean natural and/orsynthetic linear, circular and sequential arrays of nucleotides andnucleosides, e.g., cDNA, genomic DNA (gDNA), mRNA, and RNA,oligonucleotides, oligonucleosides, and derivatives thereof. The term“encoding” is intended to mean that the subject nucleic acid may betranscribed and translated into either the desired polypeptide or thesubject protein in an appropriate expression system, e.g., when thesubject nucleic acid is linked to appropriate control sequences such aspromoter and enhancer elements in a suitable vector (e.g., an expressionvector) and when the vector is introduced into an appropriate system orcell. As used herein, “polypeptide” refers to an amino acid sequencewhich comprises both full-length protein and fragments thereof.

[0029] As referred to herein, the term “capable of hybridizing underhigh stringency conditions” means annealing a strand of DNAcomplementary to the DNA of interest under highly stringent conditions.Likewise, “capable of hybridizing under low stringency conditions”refers to annealing a strand of DNA complementary to the DNA of interestunder low stringency conditions. In the present invention, hybridizingunder either high or low stringency conditions would involve hybridizinga nucleic acid sequence (e.g., the complementary sequence to SEQ ID No.1 or portion thereof, with a second target nucleic acid sequence. “Highstringency conditions” for the annealing process may involve, forexample, high temperature and/or lower salt content, which disfavorhydrogen bonding contacts among mismatched base pairs. “Low stringencyconditions” would involve lower temperature, and/or higher saltconcentration than that of high stringency conditions. Such conditionsallow for two DNA strands to anneal if substantial, as is the case amongDNA strands that code for the same protein but differ in sequence due tothe degeneracy of the genetic code. Appropriate stringency conditionswhich promote DNA hybridization, for example, 6× SSC at about 45° C.,followed by a wash of 2× SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, NY (1989), 6.31-6.3.6. For example, the salt concentrationin the wash step can be selected from a low stringency of about 2× SSCat 50° C., to a high stringency of about 0.2× SSC at 50° C. In addition,the temperature in the wash step can be increased from low stringency atroom temperature, about 22° C., to high stringency conditions, at about65° C. Other stringency parameters are described in Maniatis, T., etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring N.Y., (1982), at pp. 387-389; see alsoSambrook J. et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Volume 2, Cold Spring Harbor Laboratory Press, Cold Spring,N.Y. at pp. 8.46-8.47 (1989).

[0030] The foregoing and other aspects of the invention may be betterunderstood in connection with the following examples, which arepresented for purposes of illustration and not by way of limitation.

SPECIFIC EXAMPLE 1

[0031] Isolation and Characterization of the Cok-4 Gene

Materials and Methods

[0032] Genetic analysis of the segregating population. The bean breedingline SEL 1308 obtained from CIAT was used as the source of the Co-42gene. This line was derived from a backcross between the anthracnosesusceptible cultivar Talamanca and the recurrent parent Colorado deTeopisca (accession number G2333). SEL 1308 was crossed to Black Magic,a susceptible black bean cultivar. Hybrid seeds were advanced to the F2generation and a population of 1018 F2 individuals was developed.Progeny tests were performed in 96 F2 derived F3 families todiscriminate homozygous from heterozygous resistant genotypes. Race 73(ATCC 96512) of C. lindemuthianum was chosen to confirm the dominantinheritance of the Co-42 gene in SEL 1308. Black Magic, the susceptibleparent of the mapping population, dies in five days after inoculation.Inoculum preparation, inoculation methods, and disease characterizationof the segregating population were conducted as described by Young andKelly (Plant Dis. 80:650-654 (1996)). Individual F2 plants from BlackMagic/SEL 1308 population were screened with the SCAR marker SAS13previously found to be linked to the Co-42 gene. Procedures for SCARanalysis are described by Melotto, M. et al. (Genome 39:1216-1219(1996)). Additional RAPD markers flanking the Co-42 gene were sought byusing bulked segregant analysis (Michelmore, R. W. et al., PNAS (USA)88:9828-9832 (1991)). Inheritance of the disease phenotype and molecularmarkers was confirmed in 1018 F2 plants using the Chi-square test.Linkage analysis was performed using the Linkage-1 software (Suiter, K.A. et al., J. Hered. 74:203-204 (1983)) and distance between marker andresistance gene expressed in centiMorgan (cM) was calculated usingKosambi's function in the Linkage-1 program.

[0033] Southern analysis. Genomic DNA from bean cultivars and a BACclone was digested with EcoR I according to the manufacturer (BoehringerMannheim, Indianapolis, Ind.). Electrophoresis and blotting wereconducted using standard techniques (Sambrook, J. et al., MolecularCloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Lab. Press,Plainview, N.Y. (1989)). The COK-4 gene was labeled using the DIG HighPrime Starter kit I (Boehringer Mannheim) and used as a probe forhybridization. Stringency washes were performed in 2× SSC, 0.2% SDS and0.2× SSC, 0.1% SDS solutions. Both washes were conducted twice for 30minutes at 62° C.

[0034] Long distance PCR and primer walking. DNA clones flanking theSAS13 marker were generated using the Universal GenomeWalkerTM kit(Clontech Laboratories, Inc., Palo Alto, Calif.). DNA from SEL 1308 waspurified using phenol and chloroform, and digested with five restrictionenzyme, Dra I, EcoR V, Pvu II, Sca I, and Stu I. Adaptors were ligatedto restricted DNA samples for PCR amplification with adaptor-specificprimers. PCR reactions were carried out in 50 ul solution containing 1×Advantage Genomic Polymerase Mix (Clontech Laboratories, Inc., PaloAlto, Calif.), 1.1 mM Mg(OAc)2, 10 mM of each dNTP, 10 pM of eachadaptor-specific and SAS13 primers, and 50 ng of DNA template. PCRreactions were placed in a 9600 Thermocycler (Perkin Elmer AppliedBiosystems) and the PCR file consisted of 7 cycles of 2 seconds at 94°C., 4 minutes at 70° C., followed by 32 cycles of 2 seconds at 94° C., 4minutes at 65° C. and an extension cycle of 7 minutes at 65° C. Longdistance PCR (LD-PCR) amplification products were cloned using theTOPOTM TA Cloning kit (Invitrogen Corp., San Diego, Calif.). Bothstrands of cloned DNA fragments were sequenced using an AppliedBiosystems 377 DNA Sequencer (Perkin Elmer Applied Biosystems) aspreviously described by Melotto et al. (Genome 39:1216-1219 (1996)). Newprimers were designed based on those sequences to walk in unclonedgenomic DNA as proposed by Siebert et al. (Nucleic Acid Res.23:1087-1089 (1995)).

Results

[0035] The SCAR marker SAS13 was previously found to be tightly linkedto the Co42 gene, which conditions resistance to anthracnose in commonbean (Young, R. A. et al., Theor. Appl. Genet. 96:87-94 (1998)). Themarker co-segregated with 1014 F2 individuals in a population size of1018 (FIG. 1A). The 950-bp DNA fragment generated by the SAS13 markerwas sequenced and analyzed for similarities to sequences of knowndisease resistance genes. The alignment obtained by using BLAST searchsoftware (Altschul, S. F. et al., Nucleic Acids Res. 25:3389-3402(1997)) revealed a high similarity to serine-threonine kinase (STK)domains such as the ones encoded by the disease resistance gene Pto(gil430992; gil1809257; Martin, G. B. et al., Science 262:1432-1436(1993)) and Fen gene (gi|1098334; Martin, G. B. et al., Plant Cell6:1543-1552 (1994)) in tomato. Other proteins similar to the SAS13 DNAfragment included receptor-like kinases (RLK) from other organismsincluding Arabidopsis thaliana, Brassica sp, Oryza sativa, and Zea mays.Based on these results, the SAS13 marker was used as a starting pointfor primer walking in genomic DNA to find complete gene sequencesencoding for protein kinase domains. Four overlapping clones extendingthe original SAS13 950-bp fragment were obtained and the full length ofthe contig included 3,371 bp. The full length contig is shown in FIG.1B. The arrows indicate COK-4 specific primers and restriction sites areletter coded, B=Bam H, E=EcoR I, H=Hind II, K=Kpn I, Me=Mse I and Mp=MspI. Primer pairs were designed to test whether the generated clones werecontiguous in the plant genome. All primer sets amplified a single bandof the predicted size. Sequence analysis of the contig revealed an openreading frame (ORF) of 1110 bp, which was named COK-4 (SEQ ID NO: 1;FIGS. 1B and 1C). Two essential eukaryote promoter elements, TATA andCAAT boxes, and putative promoter sequences were found upstream of theCOK-4 gene. As shown in FIG. 2, the predicted amino acid sequence ofCOK-4 (SEQ ID NO: 3; a, FIG. 2) has a high degree of similarity withexpressed sequences generated by the Pto gene from Lycopersiconpimpinellifolium and L. esculentum (38% identity, 53% similarity and 15%gap; Martin, G. B. et al., Science 262:1432-1436 (1993)), TMK proteinfrom rice (29%, 45%, and 16%; van der Knaap, E. et al., Plant Physiol.112:1397 (1996)), extracellular S-domain from Brassica oleracea (30%,45%, 15%; PID:g2598271), S-domain receptor-like protein kinase from Z.mays (33%, 49%, 14%; PID:g3445397), and leucine-rich repeat (LRR)transmembrane protein kinase 2 from Z. mays (28%, 45%, 19%; Li, Z. etal., Plant Mol. Biol. 37:749-761 (1998)). In FIG. 2, amino acid identityis indicated by a double underline and amino acid similarity isindicated by a single underline. Numbers in the sequence indicate thefirst and last amino acids aligned. The amino acid sequences are: a)COK-4 (SEQ ID NO: 3), b) disease resistance protein kinase Pto (SEQ IDNO: 5), c) serine/threonine protein kinase Pto (Lycopersicon esculentum;SEQ ID NO: 6), d) putative serine/threonine protein kinase, Fen gene (L.esculentum; SEQ ID NO: 7), e) TMK (Oryza sativa; SEQ ID NO: 8), and f)extracellular S domain of Brassica oleracea (SEQ ID NO: 9). The proteinencoded by COK-4 was analyzed for possible functional domains. The COK-4protein has a STK domain, which includes a protein kinase ATP-bindingregion signature (amino acids 53 to 79), a primary transmembrane domain(amino acids 202 to 224), putative sites for N-myristoylation (aminoacids 7 to 12, 149 to 154, 160 to 165, 250 to 255, and 364 to 369) andN-glycosylation (amino acids 16 to 18 and 26 to 28), and a cAMP andcGMP-dependent protein kinase site (amino acids 41 to 44) (SEQ ID NO: 3;FIG. 1C). The primary transmembrane region is indicated by theunderlined amino acids (202 to 224) and the secondary transmembraneregion by the wavy underlined amino acids (226 to 248 and 256 to 278) inFIG. 1C.

Discussion

[0036] Two lines of evidence strongly suggest that the COK-4 gene,herein described, is a member of the complex Co-42 locus conditioningresistance to anthracnose in common bean. First, genetic analysisindicated co-segregation of the COK-4 gene with the resistant phenotypein a population of 1350 F3 individuals. Secondly, amino acid sequenceanalysis of the COK-4 gene, which is located 462 bp downstream of theSAS13 marker (FIG. 1B), revealed high similarity with previously clonedresistance genes and protein domains known to play an important role indisease resistance. The putative protein encoded by the COK-4 gene hasthe structure of STKs and RLKs. Receptor-like kinases contain anextracellular domain possibly functioning in ligand binding and acytoplasmic domain responsible for signal transduction (Walker, J. C.,PI. Mol. Biol. 26:1599-1609 (1994)). The COK-4 protein most likely islocalized at the membrane because it contains three highly hydrophobicregions characteristic of a transmembrane domain and has an averagehydrophobicity of −0.036 (calculated by the SOSUI software; Hirokawa, T.et al., Bioinformatics 14:378-379 (1998)). Alignment of the COK-4 aminoacid sequence with the extracellular S-domain of Brassica oleracea andLRR transmembrane and RLK domain of Z. mays also supports localizationof the COK-4 protein in the cellular membrane. If the resistance geneproduct is the receptor for the pathogen Avr gene product, it isexpected that recognition occurs at the membrane level. Colletotrichumlindemuthianum is a hemibiotrophic fungus that penetrates the bean cellwall (Bailey, J. A. et al., Infection Strategies of Colletotrichumspecies. In: Bailey, J. A., Jeger. M. J. eds. Colletotrichum—Biology,Pathology and Control. Wallington, Oxon (U.K.), CAB International 88-120(1992)). In addition, race specificity in C. lindemuthianum is expressedafter fungal penetration through the epidermal cell wall and the primaryhyphae of C. lindemuthianum remain external to the host plasma membrane,which becomes invaginated around the fungus (Bailey J. A. et al.,Infection Strategies of Colletotrichum species. In: Bailey, J. A.,Jeger. M. J. eds. Colletotrichum—Biology, Pathology and Control.Wallington, Oxon (U.K.), CAB International 88-120 (1992)). Theseobservations suggest that the avirulence gene product maybe ahost-specific elicitor located in the membrane and pathogen recognitionmay occur at the surfaces of the C. lindemuthianum infection hyphae andbean cell membrane.

[0037] Most of the disease resistance genes previously cloned conferresistance to bacterial diseases and their products are localized in thecytoplasm. For instance, Pto is soluble protein localized in thecytoplasm of tomato cells where it binds to the AvrPto protein of thebacterial pathogen (Scofield, S. R. et al., Science 2063-2065 (1996);Tang, X. et al., Science 274:2060-2063 (1996)). Bacterial Avr geneproducts are known to be secreted into the host cytoplasm through thetype III secretory system (Bent, A. F., The Plant Cell 8:1757-1771(1996)). However, little is known about the function of Avr proteinsfrom fungal pathogens and only a few fungal Avr-generated signals havebeen described. Race-specific elicitors have been partially purifiedfrom two hemibiotrophic plant pathogens, Cladosporium fulvum and C.lindemuthianum (Lamb, C. J. et al., Cell 56:215-224 (1989)). Onewell-studied example of race-cultivar specificity is the C.fulvum/Lycopersicon pathosystem. Cf proteins possess extracellulardomains, which supposedly recognize the corresponding Avr proteins(Jones, D. A. et al., Science 266:789-793 (1994); Dixon, M. S. et al.,Cell 84:451-459 (1996)). Although Avr proteins of C. lindemuthianum havenot been isolated, occurrence of race-cultivar specificity suggests thepresence of Avr-generated signal triggering plant defense response.Based on the similarities between the C. fulvum/tomato and C.lindemuthianum/bean pathosystems, one would expect that host-specificelicitors and anthracnose resistance gene products are located at themembrane where the pathogen is recognized.

SPECIFIC EXAMPLE 2 Restriction and Linkage Mapping of the Cok-4 GeneMaterials and Methods

[0038] Restriction analysis of specific PCR products. PCR primers weredesigned to amplify specific DNA fragments near the SAS13 marker region.The PCR amplification reaction contained 50 ng of genomic DNA, 10 mM ofeach dNTP, 10 pmol of each forward and reverse primers, lx enzyme buffercontaining MgSO4 and 1U of Pfu DNA polymerase (Promega, Madison, Wis.).PCR reactions were placed in a 9600 Thermocycler (Perkin Elmer AppliedBiosystems) and the PCR file consisted of 34 cycles of 20 seconds at 95°C., 30 seconds at 55° C., and 4 minutes at 72° C., followed by anextension cycle of 7 minutes at 72° C. The amplification product wasused in a digestion reaction containing 1× enzyme buffer, 10 U ofrestriction enzyme and 10 μl of PCR reaction. Digestion of DNA fragmentwas carried out for four hours at 37° C. Restriction patterns wereobserved on 0.8% ethidium bromide-stained agarose gel. TABLE I Sequenceof the specific primer set designed to amplify the COK-4 gene in thevicinity of the Co-42 locus. Primer Nucleotide position Sequence (5′-3′)Forward 405-426 GTA TGG TAA GTG ACA AGT GAG A Reverse 1578-1556 ACC TGGTCA CTT ACA TTT CTT CA

[0039] To determine the copy number of the COK-4 gene in different beancultivars, EcoR I-restricted DNA was probed with the COK-4 gene clone(FIG. 5). The restriction enzyme EcoR I does not cut the COK-4 gene. Theresistant cultivars SEL 1308 (Lane 3, FIG. 5), possessed two majorhomologous DNA sequences of 1.5 and 9 kb in size, whereas thesusceptible cultivars SEL 1360 (Lane 6, FIG. 5) and Black Magic (Lane 4,FIG. 5) possessed multiple homologous sequences of various sizes. Again,TO possessed a unique RFLP pattern with only one 9-kb DNA fragment (Lane5, FIG. 5). The BAC clone 7847 possessed at least four copies of theCOK-4 ORF. A linkage map around the COK-4 gene could be predicted usingtwo newly identified RAPD markers, OH181150 and OBB141150/1050. Thesemarkers are closely linked to the Co-42 gene and were identified byusing bulked segregant analysis (Michelmore, R. W. et al., PNAS (USA)88:9828-9832 (1991)). OH181150 is a dominant marker linked in couplingwith the Co-42 locus at 1.11 cM, whereas OBB141150/1050 is a co-dominantmarker, which amplifies a 1150-bp DNA band in the resistant parent SEL1308 and a 1050-bp DNA band in the susceptible parent Black Magic (FIG.6). The DNA of six bean cultivars was amplified with RAPD markersOBB141150/1050 (Lanes 2-7, FIG. 6) and OH181150 (Lanes 8-13, FIG. 6).The six bean cultivars were Black Magic (co-42/co-42; Lane 2, FIG. 6),SEL 1308 (Co-42/Co-42; Lane 3, FIG. 6), SEL 1360 (co42/co-42; Lane 4,FIG. 6), G2333 (Co-42/Co-42; Lane 5, FIG. 6), heterozygous resistant F2individual (Lane 6, FIG. 6), homozygous resistant F2 individual (Lane 7,FIG. 6), Black Magic (Lane 8, FIG. 6), SEL 1308 (Lane 9, FIG. 6), SEL1360 (Lane 10, FIG. 6), G2333 (Lane 11, FIG. 6), homozygous resistant F2individual (Lane 12, FIG. 6) and homozygous susceptible F2 individual(co-42/co-42; Lane 13, FIG. 6). The OBB141150/1050 marker co-segregatedwith the Co-42 gene and is linked at 2.24 cM. Linkage analysis indicatedthat these markers flank the Co-42 gene and are 4.65 cM apart. Apreviously identified marker, OAL9780 appears closely associated withOH181150 at 5.8 cM (FIG. 1A).

Discussion

[0040] Although the COK-4 region was amplified in both resistant andsusceptible parents of the mapping population, internal differences innucleotide sequences exist as indicated by restriction (FIG. 3) andsequence (FIG. 4) analyses. Single nucleotide polymorphisms (SNPs)identified in the COK-4 sequences of resistant and susceptible beanlines and co-segregation of restriction patterns with disease phenotype,indicate that the COK-4 gene is involved in anthracnose resistance.Small variation in gene sequences can result in contrasting phenotypes.In tomato, the Pto and Fen genes are present in bacterialspeck-susceptible and fenthion-sensitive genotypes and encode a proteinkinase 87 and 98% identical to the resistance alleles, respectively(Jia, Y. et al., Plant Cell 9:61-73 (1997)). A SNP found in riceaccounted for 80% of the variation in amylose content (Ayres, N. M. etal., Theor. Appl. Genet. 94:773-781 (1997)). The original SAS13 SCARmarker, which amplified a single 950-bp fragment in the resistantparent, co-segregated with the Co-42 resistance gene in the segregatingpopulation of 1018 F2 individuals. Four recombinant individuals weredetected and the distance between the marker and the Co-42 locus wasestimated at 0.39 cM. Three susceptible individuals possessing the SAS13fragment and one resistant line lacking the fragment were observed.These four F2 individuals however, possessed the COK-4 allelecorresponding to the phenotype of the plant confirmed by restrictionanalysis.

[0041] Previous genetic studies indicated that the Co-4 locus is acomplex gene family (Young, R. A. et al., Theor. Appl. Genet. 96:87-94(1998)). Two resistance alleles have been described which mapped at theCo-4 locus, one present in the bean cultivar TO and the other present inSEL 1308 as supported by allelism test and DNA sequence analysis. TOshowed a unique restriction pattern and 44 SNPs at the COK-4 regioncompared to SEL 1308. Genetic analysis indicates a single genesegregating in the Black Magic/SEL 1308 F2 mapping population, howeverother genes may be tightly clustered at the Co42 locus. Bean cultivarsappear to possess multiple copies of the COK-4 gene based on Southernanalysis. If the COK-4 homolog in TO is non-functional and differentfrom that in SEL 1308, clearly the functional Co-4 gene in TO must belinked to COK-4 and may be a gene duplication based on the RFLPpatterns. Another anthracnose resistance gene, Co-2 has also been shownto be a complex multigene family (Geffroy, V. et al., Theor. Appl.Genet. 96:494-502 (1998)). Sequence analysis of a linked marker revealedmultiple copies of LRR sequences clustered near the Co-2 gene.Resistance genes appear to be clustered in the plant genome and mayoccur in multiple copies spanning large regions of the plant genome(Kesseli, R. V. et al., Mol. PI. Microbe Interact. 6:722-728 (1993);Maisonneuve, B. et al., Theor. Appl. Genet. 89:96-104 (1994); Meyers, B.C. et al., The Plant Cell 10:1817-1832 (1998)). The RAPD markersOH181150 and OBB141150/1050 are being used to further investigate thepresence of the gene cluster and to locate the Co-4 gene in theintegrated bean map (Freyre, R. et al., Theor. Appl. Genet. 97:847-856(1998)) as they flank the entire locus.

[0042] These findings indicate that tightly linked molecular markers maybe used to identify disease resistance gene candidates. The SAS13 markertagged the Co-4 locus as it allowed the identification of differentresistance alleles present in diverse bean cultivars. The marker wasused to clone the COK-4 gene from resistant cultivars as well ashomologs present in the susceptible cultivars. The COK-4 gene thatconditions resistance to a fungal pathogen of common bean is a genehighly similar to the Pto resistance gene present in tomato. Bycomparing the COK-4 homologs, SNPs were identified and were moreaccurate than the SCAR marker in discriminating the plant genotype atthe Co-4 locus. Most important, SNPs co-segregated with the diseasephenotype in a large segregating population and could be used toidentify three different alleles at the Co-4 locus. This work representsthe first report of the successful cloning and molecularcharacterization of a disease resistance gene in grain legumes.Molecular cloning of resistance genes should facilitate studies onplant-pathogen interaction and ultimately facilitate genetic improvementof crop species.

[0043] The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingdrawings and claims, that various changes, modifications and variationscan be made therein without departing from the spirit and scope of theinvention as defined in the following claims.

[0044] All patents and other publications cited herein are expresslyincorporated by reference.

1 11 1 1110 DNA Phaseolus vulgaris 1 atgtttctga attgtgtggg catgtgttgttcgaagccca caacaaatac aacttcatct 60 cagagacagt ttccaacgtt gatagaagagctgtgccatc aattttctct caccgatctt 120 aggaaagcca ccaataactt tgatcagaagagagtaatag gaagtggatt atttagtgaa 180 gtatacaaag ggtgtctgca gcacgatggtgcttctgatt acacggtcgc aataaagcga 240 tttgattatc aaggatgggc agcgttcaacaaggaaatcg aattgctatg ccagcttcgt 300 caccctagat gtgtttctct tataggattctgcaaccacg aaaatgagaa gattcttgta 360 tacgagtaca tgtccaatgg atctctagataaacacctac aagaaggtca actatcatgg 420 aagaagaggc tggagatatg cataggagtagcacgtggac tacacttcct tcacaccgga 480 gccaagcgtt ccatctttca ctgtatcctcggtcctggta ccgtcctttt ggatgaccag 540 atggagccaa aactcgctgg tttcgatgctagcgagcagg gatcacgttt tatgtcaaag 600 cagaagcaaa tcaatgtgat cgtgttttgggtaatttttg ttttgttgta tgagctcact 660 cactgccatg attttttgtg gatcaaactaagcttactct ttgttatagg ttgtaggggc 720 tacacggcta cggactatct catggatggtatcatcacag ctaaatggga tgttttctca 780 tttggtttcc ttctactaga agttgtgtgcaggaggatgt tttatttaat aactctgact 840 aaaaaagaat gtctggagaa tcctgttgaggagagaattg atccgattat caaaggaaag 900 attgcaccag attgttggca agtgtttgtagatatgatgg taagttgctt gaagtatgaa 960 ccagatgaga gaccaacaat tggtgaagtggaggtgcaac ttgagcatgc tctatccatg 1020 caggaacaat ctgatatcac aaactccaactctgagtata ccttactctc caaaaccatt 1080 atttcccttg gagtgaagaa atgtaagtga1110 2 1090 DNA Phaseolus vulgaris 2 atgtttctga attgtgtggg catgtgttgtacgaagccca caacaaatac aacttcatct 60 cagagacagt ttccaacgtt gatagaagagctgtgccatc aattttctct ctccgatctt 120 aggaaagcca tcaataactt tgatcagaagagagtaatag gaagtggatt ttttagggaa 180 gtatattaag ggtgtctgca gcatgatggtgcttctgatt acacggtcgc aataaagcga 240 tttgattatc aaggatggga agcgttcaacaaggaaatcg aattgctatg ccagcttcgt 300 caccctagat gtgtttctct tataggattctgcaaccacc aaaatgagaa gattcttgta 360 tacgagtaca tgtccaatgg atctctagataaacacctac aagatggtga actatcatgg 420 aagaagaggc tagagatctg cataggagtagcacgtggac tacactacct tcacactggt 480 gccaagcgtt ccatctttca ctgtatcctcggtcctagta ccatcctttt ggatgaccaa 540 atggagccaa aactcgctgg tttcggtgttagcatgcagg gatcacgttt tatgtcaaag 600 cagaagcaaa tcaatgtaga tcgtgttttgggtaattttt gttttgttgt atgagctcac 660 tcactgcaat gaatttttgt ggatcaaactaagctaatac tctttgttat aggtactttt 720 ggctacccgg ctacggacta tgtcatggatggtaccatca cagctaaatg ggatgttttc 780 tcatttggtt tccttctact agaagttgtgtgcaggagga tgttttattt gataactctg 840 actaaaaaaa aatgtctgga gaatcctgttgaggagagaa ttgatccgat tatcaaaggg 900 aagattgcac cagattgttg gcaagtgtttgtagatatga tggtaacttg cttgaagtat 960 gaaccagatg agcgaccaac aattggtgaagtggaggtgc aacttgagca tgctctatcc 1020 atgcaggaac aagctgatat cacaaactccaactctgagt ataccttact gtccaaaacc 1080 attatttccc 1090 3 369 PRTPhaseolus vulgaris 3 Met Phe Leu Asn Cys Val Gly Met Cys Cys Ser Lys ProThr Thr Asn 1 5 10 15 Thr Thr Ser Ser Gln Arg Gln Phe Pro Thr Leu IleGlu Glu Leu Cys 20 25 30 His Gln Phe Ser Leu Thr Asp Leu Arg Lys Ala ThrAsn Asn Phe Asp 35 40 45 Gln Lys Arg Val Ile Gly Ser Gly Leu Phe Ser GluVal Tyr Lys Gly 50 55 60 Cys Leu Gln His Asp Gly Ala Ser Asp Tyr Thr ValAla Ile Lys Arg 65 70 75 80 Phe Asp Tyr Gln Gly Trp Ala Ala Phe Asn LysGlu Ile Glu Leu Leu 85 90 95 Cys Gln Leu Arg His Pro Arg Cys Val Ser LeuIle Gly Phe Cys Asn 100 105 110 His Glu Asn Glu Lys Ile Leu Val Tyr GluTyr Met Ser Asn Gly Ser 115 120 125 Leu Asp Lys His Leu Gln Glu Gly GlnLeu Ser Trp Lys Lys Arg Leu 130 135 140 Glu Ile Cys Ile Gly Val Ala ArgGly Leu His Phe Leu His Thr Gly 145 150 155 160 Ala Lys Arg Ser Ile PheHis Cys Ile Leu Gly Pro Gly Thr Val Leu 165 170 175 Leu Asp Asp Gln MetGlu Pro Lys Leu Ala Gly Phe Asp Ala Ser Glu 180 185 190 Gln Gly Ser ArgPhe Met Ser Lys Gln Lys Gln Ile Asn Val Ile Val 195 200 205 Phe Trp ValIle Phe Val Leu Leu Tyr Glu Leu Thr His Cys His Asp 210 215 220 Phe LeuTrp Ile Lys Leu Ser Leu Leu Phe Val Ile Gly Cys Arg Gly 225 230 235 240Tyr Thr Ala Thr Asp Tyr Leu Met Asp Gly Ile Ile Thr Ala Lys Trp 245 250255 Asp Val Phe Ser Phe Gly Phe Leu Leu Leu Glu Val Val Cys Arg Arg 260265 270 Met Phe Tyr Leu Ile Thr Leu Thr Lys Lys Glu Cys Leu Glu Asn Pro275 280 285 Val Glu Glu Arg Ile Asp Pro Ile Ile Lys Gly Lys Ile Ala ProAsp 290 295 300 Cys Trp Gln Val Phe Val Asp Met Met Val Ser Cys Leu LysTyr Glu 305 310 315 320 Pro Asp Glu Arg Pro Thr Ile Gly Glu Val Glu ValGln Leu Glu His 325 330 335 Ala Leu Ser Met Gln Glu Gln Ser Asp Ile ThrAsn Ser Asn Ser Glu 340 345 350 Tyr Thr Leu Leu Ser Lys Thr Ile Ile SerLeu Gly Val Lys Lys Cys 355 360 365 Lys 4 1111 DNA Phaseolus vulgaris 4atgtttctga attgtgtggg catgtgttgt tcgaagccca caacaaatac aacttcatct 60cagagacagt ttccaacgtt gatagaagag ctgtgccatc aattttctct caccgatctt 120aggaaagcca ccaataactt tgatcagaag agagtaatag gaagtggatt atttagtgaa 180gtatacaaag ggtgtctgca gcatgatggt gcttctgatt acacggtcgc aataaagcga 240tttgattatc aaggatgggc agcgttcaac aaggaaatcg aattgctatg ccagcttcgt 300caccctagat gtgtttctct tataggattc agcaaccacg aaaatgagaa gattcttgta 360tacgagtaca tgtccaatgg atctctagat aaacacctac aagaaggtca actatcatgg 420aagaagaggc tagagatatg cataggagta gcacgtggac tacactacct tcacaccgga 480gccaagcgtt ccatctttca ctgtatcctc ggtcctggta ccgtcctttt ggatgaccag 540atggagccaa aactcgctgg tttcggtgct agcgagcagg gatcacgttt tatgtcaaag 600cagaagcaaa tcaatgtaga tcgtgttttg ggtaattttt gtttttttgt atgagctcac 660tcactgccat gattttttgt ggatcaaact aagcttactc tttgttatag gttgttgggg 720ctacacggct acggactatc tcatggatgg tatcatcaca gctaaatggg atgttttctc 780atttggtttc cttctactag aagttgtgtg caggaggatg ttttatttga taactctgac 840taaaaaaaaa tgtctggaga atcctgttga gtagagaatt gatccgatta tcaaagggaa 900gattgcacca gattgttggc aagtgtttgt agatatgatg gtaacttgct tgaagtataa 960accagatgag agaccaacaa ttggtgaagt ggaggtgcaa cttgagcatg ctctatccat 1020gcaggaacaa gctgatatca caaactccaa ctctgagtat actttactgt ccaaaaccat 1080tatttccctg ggagtgaaga aatgtaagtg a 1111 5 310 PRT Lycopersiconesculentum 5 Thr Asn Ser Ile Asn Asp Ala Leu Ser Ser Ser Tyr Leu Val ProPhe 1 5 10 15 Glu Ser Tyr Arg Val Pro Leu Val Asp Leu Glu Glu Ala ThrAsn Asn 20 25 30 Phe Asp His Lys Phe Leu Ile Gly His Gly Val Phe Gly LysVal Tyr 35 40 45 Lys Gly Val Leu Arg Asp Gly Ala Lys Val Ala Leu Lys ArgArg Thr 50 55 60 Pro Glu Ser Ser Gln Gly Ile Glu Glu Phe Glu Thr Glu IleGlu Thr 65 70 75 80 Leu Ser Phe Cys Arg His Pro His Leu Val Ser Leu IleGly Phe Cys 85 90 95 Asp Glu Arg Asn Glu Met Ile Leu Ile Tyr Lys Tyr MetGlu Asn Gly 100 105 110 Asn Leu Lys Arg His Leu Tyr Gly Ser Asp Leu ProThr Met Ser Met 115 120 125 Ser Trp Glu Gln Arg Leu Glu Ile Cys Ile GlyAla Ala Arg Gly Leu 130 135 140 His Tyr Leu His Thr Arg Ala Ile Ile HisArg Asp Val Lys Ser Ile 145 150 155 160 Asn Ile Leu Leu Asp Glu Asn PheVal Pro Lys Ile Thr Asp Phe Gly 165 170 175 Ile Ser Lys Lys Gly Thr GluLeu Asp Gln Thr His Leu Ser Thr Val 180 185 190 Val Lys Gly Thr Leu GlyTyr Ile Asp Pro Glu Tyr Phe Ile Lys Gly 195 200 205 Arg Leu Thr Glu LysSer Asp Val Tyr Ser Phe Gly Val Val Leu Phe 210 215 220 Glu Val Leu CysAla Arg Ser Ala Ile Val Gln Ser Leu Pro Arg Glu 225 230 235 240 Met ValAsn Leu Ala Glu Trp Ala Val Glu Ser His Asn Asn Gly Gln 245 250 255 LeuGlu Gln Ile Val Asp Pro Asn Leu Ala Asp Lys Ile Arg Pro Glu 260 265 270Ser Leu Arg Lys Phe Gly Asp Thr Ala Val Lys Cys Leu Ala Leu Ser 275 280285 Ser Glu Asp Arg Pro Ser Met Gly Asp Val Leu Trp Lys Leu Glu Tyr 290295 300 Ala Leu Arg Leu Gln Glu 305 310 6 308 PRT Lycopersiconesculentum 6 Met Gly Ser Lys Tyr Ser Lys Ala Thr Asn Ser Ile Ser Asp AlaSer 1 5 10 15 Asn Ser Phe Glu Ser Tyr Arg Phe Pro Leu Glu Asp Leu GluGlu Ala 20 25 30 Thr Asn Asn Phe Asp Asp Lys Phe Phe Ile Gly Glu Gly AlaPhe Gly 35 40 45 Lys Val Tyr Lys Gly Val Leu Arg Asp Gly Thr Lys Val AlaLeu Lys 50 55 60 Arg Gln Asn Arg Asp Ser Arg Gln Gly Ile Glu Glu Phe GlyThr Glu 65 70 75 80 Ile Gly Ile Leu Ser Arg Arg Ser His Pro His Leu ValSer Leu Ile 85 90 95 Gly Tyr Cys Asp Glu Arg Asn Glu Met Val Leu Ile TyrAsp Tyr Met 100 105 110 Glu Asn Gly Asn Leu Lys Ser His Leu Thr Gly SerAsp Leu Pro Ser 115 120 125 Met Ser Trp Glu Gln Arg Leu Glu Ile Cys IleGly Ala Ala Arg Gly 130 135 140 Leu His Tyr Leu His Thr Asn Gly Val MetHis Arg Asp Val Lys Ser 145 150 155 160 Ser Asn Ile Leu Leu Asp Glu AsnPhe Val Pro Lys Ile Thr Asp Phe 165 170 175 Gly Leu Ser Lys Thr Arg ProGln Leu Tyr Gln Thr Thr Asp Val Lys 180 185 190 Gly Thr Phe Gly Tyr IleAsp Pro Glu Tyr Phe Ile Lys Gly Arg Leu 195 200 205 Thr Glu Lys Ser AspVal Tyr Ser Phe Gly Val Val Leu Phe Glu Val 210 215 220 Leu Cys Ala ArgSer Ala Met Val Gln Ser Leu Pro Arg Glu Met Val 225 230 235 240 Asn LeuAla Glu Trp Ala Val Glu Ser His Asn Asn Gly Gln Leu Glu 245 250 255 GlnIle Val Asp Pro Asn Leu Ala Asp Lys Ile Arg Pro Glu Ser Leu 260 265 270Arg Lys Phe Gly Glu Thr Ala Val Lys Cys Leu Ala Leu Ser Ser Glu 275 280285 Asp Arg Pro Ser Met Gly Asp Val Leu Trp Lys Leu Glu Tyr Ala Leu 290295 300 Arg Leu Gln Glu 305 7 286 PRT Lycopersicon esculentum 7 Tyr ArgVal Pro Phe Val Asp Leu Glu Glu Ala Thr Asn Asn Phe Asp 1 5 10 15 AspLys Phe Phe Ile Gly Glu Gly Gly Phe Gly Lys Val Tyr Arg Gly 20 25 30 ValLeu Arg Asp Gly Thr Lys Val Ala Leu Lys Lys His Lys Arg Glu 35 40 45 SerSer Gln Gly Ile Glu Glu Phe Glu Thr Glu Ile Glu Ile Leu Ser 50 55 60 PheCys Ser His Pro His Leu Val Ser Leu Ile Gly Phe Cys Asp Glu 65 70 75 80Arg Asn Glu Met Ile Leu Ile Tyr Asp Tyr Met Glu Asn Gly Asn Leu 85 90 95Lys Ser His Leu Tyr Gly Ser Asp Leu Pro Thr Met Ser Met Ser Trp 100 105110 Glu Gln Arg Leu Glu Ile Cys Ile Gly Ala Ala Arg Gly Leu His Tyr 115120 125 Leu His Thr Asn Gly Val Ile His Arg Asp Val Lys Cys Thr Asn Ile130 135 140 Leu Leu Asp Glu Asn Phe Val Pro Lys Ile Thr Asp Phe Gly IleSer 145 150 155 160 Lys Thr Met Pro Glu Leu Asp Leu Thr His Leu Ser ThrVal Val Arg 165 170 175 Gly Asn Ile Gly Tyr Ile Ala Pro Glu Tyr Ala LeuTrp Gly Gln Leu 180 185 190 Thr Glu Lys Ser Asp Val Tyr Ser Phe Gly ValVal Leu Phe Glu Val 195 200 205 Leu Cys Ala Arg Pro Ala Leu Tyr Leu SerGlu Met Met Ser Ser Asp 210 215 220 Asp Glu Thr Gln Lys Met Gly Gln LeuGlu Gln Ile Val Asp Pro Ala 225 230 235 240 Ile Ala Ala Lys Ile Arg ProGlu Ser Leu Arg Met Phe Gly Glu Thr 245 250 255 Ala Met Lys Cys Leu AlaPro Ser Ser Lys Asn Arg Pro Ser Met Gly 260 265 270 Asp Val Leu Trp LysLeu Glu Tyr Ala Leu Cys Leu Gln Glu 275 280 285 8 339 PRT Oryza sativa 8Asn Val Asn Gly Gly Ala Ala Ala Ser Glu Thr Tyr Ser Gln Ala Ser 1 5 1015 Ser Gly Pro Arg Asp Ile His Val Val Glu Thr Gly Asn Met Val Ile 20 2530 Ser Ile Gln Val Leu Arg Asn Val Thr Asn Asn Phe Ser Asp Glu Asn 35 4045 Val Leu Gly Arg Gly Gly Phe Gly Thr Val Tyr Lys Gly Glu Leu His 50 5560 Asp Gly Thr Lys Ile Ala Val Lys Arg Met Glu Ala Gly Val Met Gly 65 7075 80 Asn Lys Gly Leu Asn Glu Phe Lys Ser Glu Ile Ala Val Leu Thr Lys 8590 95 Val Arg His Arg Asn Leu Val Ser Leu Leu Gly Tyr Cys Leu Asp Gly100 105 110 Asn Glu Arg Ile Leu Val Tyr Glu Tyr Met Pro Gln Gly Thr LeuSer 115 120 125 Gln His Leu Phe Glu Trp Lys Glu His Asn Leu Arg Pro LeuGlu Trp 130 135 140 Lys Lys Arg Leu Ser Ile Ala Leu Asp Val Ala Arg GlyVal Glu Tyr 145 150 155 160 Leu His Ser Leu Ala Gln Gln Thr Phe Ile HisArg Asp Leu Lys Pro 165 170 175 Ser Asn Ile Leu Leu Gly Asp Asp Met LysAla Lys Val Ala Asp Phe 180 185 190 Gly Leu Val Arg Leu Ala Pro Ala AspGly Lys Cys Val Ser Val Glu 195 200 205 Thr Arg Leu Ala Gly Thr Phe GlyTyr Leu Ala Pro Glu Tyr Ala Val 210 215 220 Thr Gly Arg Val Thr Thr LysAla Asp Val Phe Ser Phe Gly Val Ile 225 230 235 240 Leu Met Glu Leu IleThr Gly Arg Lys Ala Leu Asp Glu Thr Gln Pro 245 250 255 Glu Asp Ser MetHis Leu Val Thr Trp Phe Arg Arg Met Gln Leu Ser 260 265 270 Lys Asp ThrPhe Gln Lys Ala Ile Asp Pro Thr Ile Asp Leu Thr Glu 275 280 285 Glu ThrLeu Ala Ser Val Ser Thr Val Ala Glu Leu Ala Gly His Cys 290 295 300 CysAla Arg Glu Pro His Gln Arg Pro Asp Met Gly His Ala Val Asn 305 310 315320 Val Leu Ser Thr Leu Ser Asp Val Trp Lys Pro Ser Asp Pro Asp Ser 325330 335 Asp Asp Ser 9 280 PRT Brassica oleracea 9 Ala Thr Asn Asn PheSer Ser Ala Asn Lys Leu Gly Arg Gly Gly Phe 1 5 10 15 Gly Thr Val TyrLys Gly Arg Leu Leu Asp Gly Lys Glu Ile Ala Val 20 25 30 Lys Arg Leu SerLys Met Ser Leu Gln Gly Thr Asp Glu Phe Lys Asn 35 40 45 Glu Val Lys LeuIle Ala Arg Leu Gln His Ile Asn Leu Val Arg Leu 50 55 60 Ile Gly Cys CysIle Asp Lys Gly Glu Lys Met Leu Ile Tyr Glu Tyr 65 70 75 80 Leu Glu AsnLeu Ser Leu Asp Ser His Ile Phe Asp Ile Thr Arg Arg 85 90 95 Ser Asn LeuAsn Trp Gln Met Arg Phe Asp Ile Thr Asn Gly Ile Ala 100 105 110 Arg GlyLeu Val Tyr Leu His Arg Asp Ser Arg Phe Met Ile Ile His 115 120 125 ArgAsp Leu Lys Ala Ser Asn Val Leu Leu Asp Lys Asn Met Thr Pro 130 135 140Lys Ile Ser Asp Phe Gly Met Ala Arg Ile Phe Gly Arg Asp Asp Ala 145 150155 160 Glu Ala Asn Thr Arg Lys Val Val Gly Thr Tyr Gly Tyr Met Ser Pro165 170 175 Glu Tyr Ala Met Asp Gly Ile Phe Ser Met Lys Ser Asp Val PheSer 180 185 190 Phe Gly Val Leu Leu Leu Glu Ile Ile Ser Gly Lys Lys AsnAsn Gly 195 200 205 Phe Tyr Asn Ser Asn Gln Asp Leu Asn Leu Leu Ala LeuVal Trp Arg 210 215 220 Lys Trp Lys Glu Gly Lys Trp Leu Glu Ile Leu AspPro Ile Ile Ile 225 230 235 240 Asp Ser Ser Ser Ser Thr Gly Gln Ala HisGlu Ile Leu Arg Cys Ile 245 250 255 Gln Ile Gly Leu Leu Cys Val Gln GluArg Ala Glu Asp Arg Pro Val 260 265 270 Met Ala Ser Val Met Val Met Ile275 280 10 22 DNA Artificial Sequence Description of ArtificialSequencesynthetic oligonucleotide primer 10 gtatggtaag tgacaagtga ga 2211 23 DNA Artificial Sequence Description of ArtificialSequencesynthetic oligonucleotide primer 11 acctggtcac ttacatttct tca 23

We claim:
 1. An isolated nucleic acid comprising the nucleotide sequenceof SEQ. ID. No.
 1. 2. An isolated nucleic acid comprising the nucleotidesequence of SEQ. ID. NO.
 2. 3. The isolated nucleic acid of claims 1 or2, wherein the nucleotide sequence encodes the polypeptide of SEQ. ID.NO.
 3. 4. A vector comprising the nucleic acid of claim
 1. 5. A hostcell comprising the vector of claim
 4. 6. The host cell of claim 5,wherein the host cell is a plant cell.
 7. A vector comprising thenucleic acid of claim
 2. 8. A host cell comprising the vector of claim7.
 9. The host cell of claim 8, wherein the host cell is a plant cell.10. An isolated nucleic acid comprising a nucleotide sequence capable ofhybridizing under high stringency conditions to SEQ. ID. NO. 1 or thecomplement of SEQ. ID. NO.
 1. 11. The nucleic acid of claim 10, whereinthe nucleic acid is capable of conferring disease resistance to a plantcell.
 12. A vector comprising the nucleic acid of claim
 10. 13. A hostcell comprising the vector of claim
 12. 14. The host cell of claim 13,wherein the host cell is a plant cell.
 15. A method for conferringdisease resistance to a plant comprising introducing a nucleic acidcomprising SEQ. ID. NO. 1 or SEQ. ID. NO. 2 into a plant.
 16. The methodof claim 15, wherein the plant is a common bean plant of the familyPhaseolus vulgaris.
 17. The plant of claim 16, wherein the plant is adry bean plant.
 18. The method of claim 15, wherein the disease is aplant fungal disease.
 19. The disease of claim 18, wherein the diseaseis anthracnose.
 20. A transgenic plant produced by the method of claim15.
 21. The seeds of the plant of claim
 20. 22. A transgenic plantproduced by breeding the plant of claim 20, wherein the plant retainsthe trait of disease resistance.
 23. The seeds of the plant of claim 22.24. A method for conferring disease resistance to a plant comprisingintroducing a nucleic acid encoding for COK-4, wherein the nucleic acidhas one restriction site for a restriction enzyme selected from thegroup consisting of Kpn I, Mse I and Msp I.
 25. A transgenic plantproduced by the method of claim
 24. 26. The seeds of the plant of claim25.
 27. A transgenic plant produced by breeding the plant of claim 25,wherein the plant retains the trait of disease resistance.
 28. The seedsof the plant of claim 27.